Immunoassays are becoming increasingly popular as methods for detecting or monitoring the presence of drugs or analytes in body fluids or biological samples. A particular challenge in the development of immunoassays is the production of an antibody to the target drug/analyte since many are not inherently antigenic. Generally, the drug must be modified to make an antigenic derivative, yet the antibody produced to the antigenic derivative must be able to recognize the drug as it is contained in the fluid specimen to be tested with an appropriately useful degree of sensitivity, generally a level with physiological and/or pharmacological significance. Sensitivity is not the only concern. Often a variety of metabolites exist and other drugs may be present along with the target analyte. Preferably, an antibody to a particular drug or metabolite has minimal, if any, cross-reactivity with other metabolites or other drugs.
Lateral flow tests, also known as Lateral Flow Immunochromatographic Assays, are devices intended to detect the presence (or absence) of a target metabolite in sample (matrix) without the need for specialized and costly equipment, though many lab based applications exist that are supported by reading equipment. Typically, these tests are used for medical diagnostics either for home testing, point of care testing, or laboratory use, as well as law enforcement testing of drivers of vehicles.
The technology is based on a series of capillary beds, such as layers of porous paper or sintered polymer. Each of these layers has the capacity to transport fluid (e.g., urine) spontaneously. A first layer, (the sample pad) acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid migrates to the second layer (conjugate pad) in which the manufacturer has stored the so-called conjugate, a dried format of bio-active compounds (see below) in a salt-sugar matrix that contains a complete reagent mixture needed to guarantee an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner (e.g., a primary antibody) (optionally a second, labeled antibody (secondary antibody) (e.g., sandwich assay) that has been immobilized on the particle's surface. While the sample fluid dissolves the salt-sugar matrix, it also dissolves the reagents and in one combined transport action the sample and reagents mix while flowing through the porous structure. In this way, the metabolite binds to the antibodies while migrating further through the third capillary bed. This bed has one or more areas (often called stripes) where a third molecule (usually a labeled antibody) has been immobilized by the manufacturer. By the time the sample-conjugate mix reaches these stripes, the metabolite has been bound on the first antibody and the third ‘capture’ molecule binds the complex. After a while, when more and more fluid has passed the stripes, antibody/metabolite complex accumulate and the striped-area changes color. Typically there are at least two stripes: one (the control) that captures any particle and thereby shows that reaction conditions and technology worked. The second striped-area contains a specific capture molecule and only captures those complexes comprising the metabolite molecule and the first antibody. After passing these reaction zones, the fluid enters the final porous material, the wick, that provides the oncotic pressure that draws the test fluid through the multiple capillary beds and acts as a waste container.
Lateral Flow Tests can operate as either competitive or sandwich assays. In a competitive assay, the complex is rinsed, the antibody is limited or there is a target analog that competes with the target for primary antibody binding. In a sandwich assay, a primary antibody is used which is specific to the antigen and a secondary antibody is located in the third layer, the secondary antibody specific only to the first antibody. When a secondary antibody is used, it may be conjugated to a visual (or visualizable) label such as a fluorophor or an enzyme specific to a visualized chromophor.
Ethyl glucuronide (“EtG”), which is shown in FIG. 3, is a direct metabolite of ethyl alcohol formed by the conjugation of ethanol with activated glucuronic acid in the presence of UDP glucuronyl transferase on mitochondrial membranes. While ethanol is detectable for only a few hours after consumption, EtG is detectable for up to about four or five days after alcohol consumption, making it a reliable target analyte for determining alcohol consumption, assuming that the test can be validated and controlled for false positive and false negatives. Monitoring of alcohol consumption is important for many reasons including use in conjunction with alcohol abuse and abstinence testing and monitoring (for one or more of medical treatment, and court or law enforcement testing and monitoring), safety-sensitive programs such as airline pilots and health care or emergency care professionals, as well as operators of heavy machinery or vehicles.
Currently, EtG is accurately and reproducibly detected by gas chromatography/mass spectrometry (“GC/MS”) and liquid chromatography/tandem mass spectrometry (“LC/MS/MS”). An enzyme linked immunoassay (“ELISA”) test to detect EtG using a polyclonal antiserum has been attempted, but the use of polyclonal antibodies can result in an significant and increased number of false positives and false negatives, which indicates poor specificity, and sensitivity of the assay. Additionally, exposure to external ethanol (e.g., personal care products, first aid products, cleaning products, and the like that contain ethanol also produces false positives. This inaccuracy has led to recommendations or policies that only high levels of EtG (e.g., at least 500, 750, or 1000 ng/mL of urine) are acceptable for a positive test result, which can result in a significant number of false negatives, especially using immunoassay testing for EtG. Accordingly, no product appears to be on the market to provide the advantages of an accurate, reproducible, and commercially viable immunoassay for EtG, that optionally does not require further validation by the use of additional testing including (“GC/MS”) and liquid chromatography/tandem mass spectrometry (“LC/MS/MS”). Such tests also require a significant amount of time to provide results, e.g., at least hours, days, or weeks.
Therefore, there is a need or provide and solve one or more of the current problems related to developing devices and methods for an accurate, reproducible, and commercially viable immunoassay for EtG, e.g., that provides one or more of better sensitivity and/or specificity (e.g., with the use of EtG-specific monoclonal antibodies), lower and/or acceptable rates of false negatives and/or false positives, that does not require a second round of testing to confirm positive test using a much more expensive and time consuming and laboratory required test (e.g., GC/MS and/or LC/MS/MS), as compared to prior assays. Accordingly, there is also a need for systems and methods of making that provide improvements over known systems or methods that optionally overcome one or more of these problems.